The present invention relates to a novel compound and a method of analyzing a hydroxy-containing compound with said novel compound being used as a labelling agent for fluorescence.
There are many compounds that contain a hydroxyl group. They exist in the human body in such forms as adrenocortical hormones, membrane components, bile acid, sex hormones, prostaglandins, and vitamins, and they are also available as natural and synthetic alcohols, medicines, perfumes, etc. Illustrative compounds that contain primary and secondary hydroxyl groups are corticosterone, and aldosterone; illustrative compounds that contain a secondary hydroxyl group are cholesterol, colic acid, .beta.-estradiol, prostaglandin E.sub.2 and ergocalciferol; compounds that contain secondary and tertiary hydroxyl groups include 2,5-hydroxycholecalciferol. Exemplary naturally occurring substances and synthetic medicines that contain a hydroxyl group include benzyl alcohol and lauryl alcohol which have a primary hydroxyl group; mephenesin having primary and secondary hydroxyl groups; cyclohexanol and propranolol which have a secondary hydroxyl group; and ecdysones having secondary and tertiary hydroxyl groups. Many of these compounds exhibit physiological activities in vivo in trace amounts, and some of the naturally occurring substances and synthetic medicines do so such as to produce toxicity. Analysis of these hydroxy-containing compounds will be useful not only in medical and biochemical studies but also in clinical and environmental testing applications. For this purpose, great benefit would be offered by a method that is capable of high-sensitivity analyzing of hydroxy-containing compounds in a sample of interest by labelling them for fluorescence. Many compounds have so far been proposed for use as reagents that can label the hydroxyl group for fluorescence, and they include: 4-dimethylamino-1-naphthoylnitrile (Chem. Pharm. Bull, 29 (3), 899-901 (1981)), 1-anthroylnitrile (Analytica Chimica. Acta, 147, 397-400 (1983)), 7-methoxycoumarin-3-carbonyl azide (Chem. Pharm. Bull, 33 (3), 1164-1169 (1985)), and naphthyl isocyanate (J. Chromatogr., 237, 399-406 (1982)).
These reagents are used in analyzing compounds having primary and secondary hydroxy groups in the following manner: a mixture of the reagent and a sample of interest is held at a temperature ranging from room temperature to 100.degree. C. for a period of from 30 minutes to 2 hours until the sample is labelled for fluorescence, and the labelled sample is then subjected to high-performance liquid chromatography. The detection limits of these reagents are within the range of from 0.1 to 1 picomoles. The most sensitive 7-methoxycoumarin-3-carbonyl azide achieves a detection limit of 3 fentomoles for cholesterol; however, the intensity of fluorescence has a linear relationship with the amount of the sample in a very narrow range of 3 to 40 fentomoles and considerable skill is therefore required to achieve quantitative analyses. Naphthyl isocyanate has been used in analyzing steroid compounds having a tertiary hydroxyl group by performing reaction at 140.degree. C. for a period of 120 minutes but the detection limit achieved is not very high and is within the range of 2.5 to 50 picomoles.
Under these circumstances, it has been desired to develop a reagent capable of high-sensitivity labelling for fluorescence that reacts quantitatively with each of primary, secondary and tertiary hydroxyl groups to provide for easy labelling for fluorescence and which ensures quantitative analyzing over a wide detection range.